Bacillus Strains as Effective Biocontrol Agents Against Phytopathogenic Bacteria and Promoters of Plant Growth

Modern crop production relies on the application of chemical pesticides and fertilizers causing environmental and economic challenges. In response, less environmentally impactful alternatives have emerged such as the use of beneficial microorganisms. These microorganisms, particularly plant growth-promoting bacteria (PGPB), have demonstrated their ability to enhance plant growth, protect against various stresses, and reduce the need for chemical inputs. Among the PGPB, Bacillus species have garnered attention due to their adaptability and commercial potential. Recent reports have highlighted Bacillus strains as biocontrol agents against phytopathogenic bacteria while concurrently promoting plant growth. We also examined Bacillus plant growth-promoting abilities in Arabidopsis thaliana seedlings. In this study, we assessed the potential of various Bacillus strains to control diverse phytopathogenic bacteria and inhibit quorum sensing using Chromobacterium violaceum as a model system. In conclusion, our results suggest that bacteria of the genus Bacillus hold significant potential for biotechnological applications. This includes developments aimed at reducing agrochemical use, promoting sustainable agriculture, and enhancing crop yield and protection. Supplementary Information The online version contains supplementary material available at 10.1007/s00248-024-02384-1.


Introduction
Crop production relies on the application of chemical pesticides, fertilizers, and other inputs to protect plants from pathogens and increase yield [1][2][3][4].Recently, there has been a development of less environmentally impactful alternatives.Beneficial microorganisms have been demonstrated to effectively promote plant growth and development while providing protection against both biotic and abiotic stress [5].Among these soil bacteria that offer benefits to plants are the genera Acetobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Klebsiella, Pseudomonas, and Serratia, collectively termed as plant growth-promoting bacteria (PGPB) [6,7].Pseudomonas and Bacillus are the most prevalent genera, attributed to their adaptability to various environmental conditions [8].Notably, biological products based on Bacillus strains have exhibited the potential to enhance crop yields by up to 40% [8].Among the commercially utilized Bacillus strains, B. subtilis and B. amyloliquefaciens stand out [9].
Furthermore, there is evidence to suggest that free-living microorganisms in the rhizosphere modulate plant auxin and cytokinin signaling pathways, subsequently leading to alterations in root architecture and improved nutrient absorption.The Bacillus genus encompasses soil isolates that can enhance plant growth by increasing auxin and cytokinin levels and modifying root structure [10].Additionally, B. thuringiensis (Bt) and B. cereus produce insecticidal crystalline proteins (Cry) during sporulation.Utilizing these proteins can reduce the reliance on chemical insecticides, lower production costs, and minimize environmental impact, thereby promoting a more sustainable and economically viable agricultural model [10,11].
On the other hand, selected PGPBs protect plants from a range of pathogens and pests, including bacteria, fungi, viruses, and nematodes [12][13][14] while the precise mechanisms underlying this protection are not yet fully understood.Bacteria have the ability to communicate each other through production of signal molecules as N-acyl-homoserine lactones (AHLs) depending on cell density.This process has been named as quorum sensing (QS) modulating diverse functions as growth, biofilm formation, and virulence factor production [15].Besides, bacteria have the ability to modulate virulence through the production of lactonases.These enzymes can degrade the QS signals, a process known as quorum quenching (QQ).In this context, the aiiA gene, which encodes a lactonase capable of degrading AHLs, has been identified in several Bacillus strains, acting as quorum sensing inhibitors (QSI) or quorum quenchers [16].Indeed, the application of a Bacillus spp.lysate to a Pseudomonas aeruginosa culture resulted in a remarkable 93% inhibition of biofilm formation [17].
In this study, we evaluated different Bt strains as potential biocontrol agents against three phytopathogenic bacteria.We also assessed their QS inhibition activity using the Chromobacterium violaceum bacteria, a natural indicator strain that produces violacein, a pigment which production by C. violaceum is under control of the QS system.Furthermore, we examined the plant growth-promoting and developmental abilities of Bacillus strains.Our results have significant implications for the development of biotechnological applications aimed at reducing the use of persistent agrochemical products in agriculture, thereby enhancing crop yield and protection in a sustainable manner.

Bacterial Strains and Growth Conditions
Fourteen different Bt strains were employed.Ten of them were obtained from the National Collection of Microbial Strains and Cell Cultures (CDBB) of Cinvestav, and four from our laboratory strain collection (Table 1).Chromobacterium violaceum was provided by the Environmental Microbiology and Phytopathology Laboratory from the Institute of Ecology (Inecol).Clavibacter michiganensis, Ralstonia solanacearum, and Xanthomonas campestris were kindly provided by the National Phytosanitary Reference Center (CNRF), Mexico.All bacterial strains were activated and routinely grown in Luria Bertani (LB) medium and incubated in a shaker at 120 rpm and 28 °C, otherwise indicated.

Detection of the AHL Lactonase (aiiA) Gene in Bt Strains
To investigate the presence of the AHL lactonase (aiiA) gene in Bt strains, we performed the following steps: Genomic DNA from Bt strains was isolated following the method described by [18].The purity and concentration of the extracted DNA was determined by a NanoDrop One spectrophotometer (Thermo Scientific, Waltham MA, USA), and DNA integrity was verified by agarose gel electrophoresis.Amplification of the aiiA gene was performed using the forward and reverse specific primers: aiiAF (5′-ATG GGA TCC ATG ACA GTA AAG AAG CTT TAT -3′) and aiiAR (5′-GTC GAA TTC CTC AAC AAG ATA CTC CTA ATG -3′), respectively [19].PCR was carried out using 5 ng of genomic DNA and Platinum Taq DNA polymerase High Fidelity (Invitrogen, Carlsbad CA, USA) commercial system following the manufacturer's instructions.The reaction conditions were 94 °C for 1 min, 30 cycles at 94 °C for 10 s, 54 °C for 30 s, and 72 °C for 50 s, with a final extension at 72 °C for 3 min.16s rRNA gene was used as endogenous control using the forward and reverse specific primers: Bakt_341F (5′-CCT ACG GGNGGC WGC AG-3′) and Bakt_805R (5′-GAC TAC HVGGG TAT CTA ATC C-3′), respectively [20].PCR conditions were 94 °C for 1 min, 30 cycles at 94 °C for 10s, 52 °C for 30s, and 72 °C 50s, with a final extension at 72 °C for 3 min.The amplified products were then confirmed through agarose gel electrophoresis.
The Bt sequences of the aiiA gene were translated using EMBOSS Transeq (https:// www.ebi.ac.uk/ Tools/ st/ emboss_ trans eq/, accessed 05 July 2023) with bacterial codon usage.The sequences used for the construction of the phylogenetic tree were those obtained in this work and those used by Noor et al. [22].The obtained sequences were aligned with MUS-CLE using MEGA X.The phylogenetic tree was constructed based on the amino acid sequences with the neighbor-joining method using 1000 bootstrap replicates.

Confrontations of Bt Strains Against Phytopathogenic Bacteria
Bt strains which amplified for the lactonase gene were grown in liquid LB medium 24 h before the confrontation assay.Each pathogenic bacterium was grown in LB medium, X. campestris (24 h), C. michiganensis (48 h), and R. solanacearum (72 h) and then mixed with soft agar (1%) at a final bacterial concentration of 30% (approximately 2.4 × 10 5 cells/mL).This soft agar mixture was laid over a solid LB base.Subsequently, different volumes (5, 10, and 20 µL) of Bt cultures of approximately 2.4 × 10 5 cells/mL were added.
After incubation at 28 °C for 48 h, the growth inhibition halos were measured.Three independent replicates were performed for each assay.

Quorum Sensing Inhibition Assays
Quorum sensing (QS) inhibition was screened and evaluated by confronting Bt strains or Bt extracts with Chromobacterium violaceum bacteria.For QS inhibition screening, LB medium containing C. violaceum at a final concentration of OD600 = 0.1 (optical density at 600 nm) was poured onto Petri dishes and allowed to solidify.Subsequently, 1.2 × 10 4 cells/mL of each Bt strain culture were spotted over C. violaceum and LB plates (as growth control).After an incubation at 28 °C for 48 h, inhibition halos were observed.For QS inhibition evaluation, five Bt strains which produced inhibition halos over C. violaceum plates were grown in 150 mL of LB for 48 h.Cultures were centrifuged at 9000 rpm at 4 °C for 10 min.To obtain Bt extracts, cell culture supernatants were extracted with 1 volume of sodium acetate.The organic extracts were rotary evaporated at 45 °C and 250 mbar in a R-100 rotavapor (Büchi, Laguna Hills, CA) and resuspended in methanol at a final concentration of 26.8 mg/mL.For the QS inhibition assay, solid LB containing OD 600 = 0.1 of C. violaceum was prepared and 15 µL of Bt extracts was spotted.After incubation at 28 °C for 72 h, QS was evaluated through the measurement of inhibition halos.Three replicates were used for each assay.

Plant Growth Promotion Assay
To evaluate potential effects of Bt strains on in vitro co-cultivated plants, Arabidopsis thaliana (Col-0) and transgenic lines ARR5::uidA [23] and DR5::uidA [24] transgenic lines were used.These recombinant lines are used as cytokinin and auxin indicators, respectively.Arabidopsis seeds were surface sterilized as described [25] and grown in Murashige and Skoog (MS) medium in a growth chamber at 26 °C, under a 16 h/8 h light/dark photoperiod and 60% relative humidity.Bt strains were grown in solid LB (24 h) and inoculated by streaking on plates containing MS medium.
Immediately, 6-day-old Arabidopsis seedlings were placed on the bacterial streak.Co-cultures were grown for a further 10-day period in the growth chamber.Three replicates were used for each assay.Different growth parameters such as primary root length (cm) and lateral root number and histochemical GUS activity were determined and compared to control seedlings without bacterial strains.

Histochemical Determination of Reporter Beta-Glucuronidase (GUS) Activity
The reporter gene uidA gene of Escherichia coli, encoding the enzyme β-glucuronidase (GUS), was employed in the present study.To detect its activity, transgenic seedlings were immersed in a solution of 5-bromo-4-chloro-3indolyl-β-D-glucuronide (X-Gluc) in 100 mM (pH 7) sodium phosphate supplemented with 2 mM H 4 Fe(CN) 6 and 2 mM K 3 Fe(CN) 6 at 37 °C and incubated overnight.Tissue clearing was performed by incubating the seedlings in 2 mL solution of 20% (v/v) methanol and 0.24 N HCl at 60 °C for 45 min.After the removal of the solution, 7% NaOH was added to cover the seedlings and maintained at room temperature for 30 min.The solution was discarded and the seedlings were washed with 40%, 20%, and 10% (v/v) ethanol for 30 min each, and finally stored with 50% glycerol (v/v) at 4 °C [26].Plant tissues were fixed on glass slides with 50% (v/v) glycerol and representative images were taken using Nomarski optics in a Leica DM5000B microscope at 20× magnifications.

Statistical Analysis
Ten plants for each treatment were employed, assayed in three replicates.To identify differences between multiple groups' means while controlling the experiment-wise error rate, the data obtained were statistically analyzed by average comparison and one-way ANOVA (analysis of variance) with Tukey's post hoc test for multiple comparisons using SAS software and graphed with the GraphPad Prism v. 8.0.

Detection and Phylogenetic Analysis of the AHL Lactonase (aiiA) Gene in Bt Strains
While it is known that Bt produces various antimicrobial molecules such as bacteriocins, lipopeptides, and cell wall hydrolytic enzymes [27], our study aimed to investigate if Bt strains could disrupt the quorum sensing (QS) of phytopathogenic bacteria.Therefore, we first tried to detect in the Bt strains the lactonase encoding gene (aiiA), an enzyme that catalyzes the lactone-ring opening of AHL inactivating the QS signal and production of virulence factors.A 750-bp PCR product was amplified from 11 out of the 14 Bt strains analyzed (Fig. 1A).The aiiA gene could not be amplified in Bt R3, Bt strain 1, and Bt strain 2 using the specified primers, warranting further investigation into the reasons for this lack of amplification.However, amplified aiiA gene from the other 11 Bt strains were cloned and sequenced to confirm their identity.The obtained sequences were deposited in the GenBank database under the accession numbers OR271315 to OR271325.We conducted a phylogenetic analysis using genes encoding Bacillus lactonases (Fig. 1B) to understand the relationships and similarities among different strains.Eight Bacillus strains (OR271323, OR271325, OR271324, OR271320, OR271319, OR271316, OR271317, and OR271318) were clustered with Bt serovar kurstaki, indiana, and aizawai lactonases, demonstrating high similarity, while Bt serovar kenyae (ORF271315) was more distant but derived from the same node.On the other hand, the lactonase sequence OR271322 was clustered with Bt serovar entomocidus and Bt serovar alesti (OR271321) was situated on an external branch.

Bt Strains Inhibited Phytopathogenic Bacterial Growth
The 11 Bt strains in which aiiA gene was amplified were tested for their capacity to inhibit the growth of phytopathogenic bacteria (including X. campestris, R. solanacearum, and C. michiganensis).Growth inhibition halos were measured in Petri dishes from the confrontation assays.All Bt strains showed apparent inhibition activity against phytopathogenic bacteria, some of them in a concentrationdependent manner (Fig. 2).Bt serovar alesti showed the highest inhibition against C. michiganensis, followed by Bt serovar kurstaki strain 2 and Bt R4 (Fig. 2A).In the case of R. solanacearum, Bt R4 exhibited the best results inhibiting this phytopathogenic bacteria followed by Bt serovar alesti and Bt serovar kurstaki strain 2 (Fig. 2B).Finally, in the confrontation assay with X. campestris, the highest inhibition of growth was observed with Bt serovar alesti followed by Bt R4 and Bt R2 (Fig. 2C).

Bt Strains Disrupted Quorum Sensing
Bt strains in which aiiA gene was amplified were also tested for their ability to disrupt QS.For this purpose, C. violaceum was used as the accumulation of the compound violacein occurs when bacterial population reaches a high density [28].For QS inhibition screening, 11 Bt strains were tested and five of them were selected based on the presence of inhibition halos (Table S1).Then, the extracts of these Bt strains were assayed for their capacity to inhibit violacein synthesis.
The halos representing the absence of violacein were quantified to determine if the extracts, containing lactonase, disrupted C. violaceum QS as described in [28].
All the Bt strain extracts inhibited violacein synthesis, although Bt R1 produced the largest inhibition halos compared to the purified lactonase (positive control) (Fig. 3).Statistical analysis of QS inhibition evaluated by triplicate formed a group with the positive control and Bt R1, followed by Bt tolworthi, which showed 74% inhibition of violacein synthesis, compared to the negative control with methanol.Bt kurstaki strain 1, Bt kenyae, and Bt alesti isolates grouped together showing from 49 to 60% QS inhibition.

Bt Strains Promoted Plant Growth
To evaluate the ability of Bt strains in the plant growth promotion, Arabidopsis seedlings were used as an experimental model.Six-day old Arabidopsis seedlings were coinoculated on a bacterial streak and growth further ten days.Primary root length and lateral secondary root number were determined for each treatment (Fig. 4).The root architecture in all treatments consisted of a main root and lateral roots with different number and length.Likewise, the development of normal photosynthetic cotyledonary leaves was observed (Fig. 4A).Statistical analysis showed that the treatments were clustered in five groups based on the effect on primary root growth and architecture.Bt entomocidus and Bt R2 induced primary root growth with statistical significance compared with the control.On the other hand, there was a statistically significant decrease in the primary root length with R4.The other Bt strains did not show statistical differences compared with the control treatment (Fig. 4B).For secondary root number, Bt R4 produced the highest amount followed by Bt entomocidus, Bt R2, and Bt R1.The other Bt strains performed similar to the control treatment (Fig. 4C).
In a parallel assay, Arabidopsis seedlings were co-inoculated with pathogenic bacteria (Fig. S1).Interestingly, X. campestris and C. michiganensis increased primary root length and induced formation of secondary roots with statistical significance, comparable to some Bt strains.In contrast, R. solanacearum failed to induce primary root growth, but significantly increased secondary root number.

Bt Strains Induced the Expression of Auxin and Cytokinin Responses in Arabidopsis Seedlings
As shown, plant-bacteria interaction resulted in modifications to root architecture, which may be mediated by auxins and cytokinins.To explore this possibility, we assayed Arabidopsis line reporter seedlings harboring the synthetic DR5 auxin responsive element directing the expression of the GUS reporter gene [24], and a second Arabidopsis expressing the GUS reporter gen under the control of the ARR5 pseudo-response regulator gene promoter responsive to cytokinins [29].Increased accumulation of auxins and cytokinins was monitored by histochemical GUS staining of seedlings treated with different Bt strains (Fig. 5).Bt R1, Bt R2, and Bt alesti treatments led to the highest number of root hairs, which directly contribute to nutrient absorption (Fig. 5A).Plant-bacterial interactions with DR5::uidA showed higher levels of auxin accumulation after treatment with Bt R1, Bt R2, Bt tolworthi, Bt alesti, Bt kurstaki strain 1, and Bt kurstaki strain 2 (Fig. 5B).The accumulation of auxins in leaf tissue was observed in the interactions with Bt R1, Bt R2, Bt kenyae, and Bt entomocidus.In contrast, auxin accumulation was undetectable in leaves of control plants, and only observed in the apical region of the primary root.
On the other hand, transgenic line ARR5::uidA seedlings with Bt R2, Bt kurstaki strain 1, and Bt entomocidus induced cytokinin accumulation in apical roots, stems, and leaves (Fig. 5C).Of note is the differential cytokinin accumulation in leaves of the treated plants, which showed a patchy distribution, in contrast to control plant tissue which showed a uniform accumulation pattern of the reporter gene.Parallel experiments were conducted with both plant transgenic lines and pathogenic bacteria (Fig. S2).The strongest auxin induction was observed with R. solanacearum, in which the Arabidopsis seedling developed thickened roots, as well as a greater number of hairy roots displaying strong GUS expression.The accumulation of auxins in leaf tissue was observed in the interaction with C. michiganensis and R. solanacearum (Fig. S2).

Discussion
The transition towards sustainable agriculture has amplified the interest in exploring alternatives to chemical fertilizers and pesticides.Plant bio-stimulating microorganisms encompass bacteria that either facilitate plant growth and development or protect crops against pests and diseases [30].Among these, numerous bacterial species within the Bacillus genus are recognized as PGPB.
The mechanisms governing PGPB activity involve the production of plant growth regulators, enhanced nutrient   uptake from the soil, and the secretion of deterrent facagainst phytopathogenic bacteria [31].Notably, Bacillus produce a variety of antimicrobial agents, such as bacteriocins and defensins, which exhibit effectiveness against a broad spectrum of gram-negative bacteria [32].Conversely, QS is a mechanism that enables bacteria to adapt to environmental stress, including extreme temperatures and nutrient scarcity.QS is also associated with bacterial virulence, flagella synthesis, aerobic respiration, resistance to toxins and oxidative stress, biofilm formation, and the production of toxins, effector molecules, extracellular enzymes, and polysaccharides [22,33].Furthermore, the ability to disrupt QS, known as QQ, is a widespread strategy employed by bacteria across various domains of life, allowing them to compete with other bacteria and deter bacterial infections in eukaryotes.Inhibition of QS in phytopathogenic bacteria may offer a highly effective alternative for disease control.Bacillus, in particular B. thuringiensis, has been reported to produce insecticidal proteins [34], as well as antibacterial compounds and lactonases that are instrumental in restricting growth of gram-negative bacteria by hydrolyzing the diffusible signals that govern QS [35].
To assess the potential QS inhibition activity in B. thuringiensis strains, we initially attempted to amplify the lactonase gene from bacterial genomic DNA.The identification of Bt strains harboring sequences (ORF271315 to ORF271325) bearing a high similarity to aiiA genes encoding lactonases suggested their potential to disrupt QS.Phylogenetic analysis indicates a high degree of homology among lactonases in several Bacillus strains, implying a conserved role.This notion is reinforced by studies revealing that a mutated lactonase (H109Y) exhibits reduced activity compared to wild-type lactonases, highlighting a degree of specialization in Bacillus QS disruption [22,35].
Subsequently, 11 Bt strains in which the aiiA gene was amplified were tested for their ability to inhibit phytopathogenic bacteria, specifically R. solanacearum, X. campestris, and C. michiganensis.The selection of these strains was based on their demonstrated pathogenicity and virulence.R. solanacearum infects over 200 plant species worldwide, including potato (Solanum tuberosum), tomato (Solanum lycopersicum), and banana (Musa spp.) [17,36].Xanthomonas spp.infect approximately 400 plant species, including sugar cane, beans, cassava, cabbage, banana, citrus, tomato, chili pepper, and rice [37].Their life cycle involves epiphytic and endophytic stages, with the latter marked by biofilm formation in the plant's vascular system, enhancing their pathogenicity [38,39].C. michiganensis is a gram-positive bacterium that infects the xylem of plants and causes significant agricultural losses [40].Notably, C. michiganensis regulates its QS via cyclic peptides (oligopeptides) [41,42], suggesting that Bacillus-mediated QQ for this bacterium occurs through secreted proteases targeting these peptide signals.
C. violaceum, which serves as a model for direct QSmediated violacein synthesis inhibition [21,28], was used to screen and evaluate the inhibition of QS by Bt strains.The statistical analysis revealed that the selected strains effectively inhibited QS.Bt R1 and Bt tolworthi displayed 90% and 74% inhibition, respectively, while Bt kurstaki strain 1, Bt kenyae, and Bt alesti exhibited inhibition percentages ranging from 49 to 60%.These results suggest that Bt strains have the potential to mitigate damage to agricultural crops caused by these pathogenic bacteria.The inactivation of genes associated with QS (rpfF, rpfC, and RpfG) significantly reduced the virulence of Xanthomonas citri, which infects grapefruit leaves [43].Conversely, overexpressing the Xylella fastidiosa rpfF gene in Carrizo citrange plants intensified the damage caused by X. citri at inoculation sites, indicating that heterologous QS signals disrupt signaling [44].These findings underscore the role of QS in pathogenicity and virulence during infection and lay the foundation for using QS inhibition and QQ to control phytopathogenic bacteria.
Regarding the assessment of Bt strains as PGPB, Bt entomocidus and Bt R2 were found to promote growth and development in Arabidopsis seedlings, particularly in terms of primary root length.Bt R4, on the other hand, resulted in the highest number of secondary roots, followed by Bt entomocidus, Bt R2, and Bt R1.Additionally, Bt R1, Bt R2, and Bt alesti treatments led to the highest number of root hairs, which directly contribute to nutrient absorption.These findings align with previous reports on the advantages of the use Bacillus as PGPB and microbial plant biostimulants (MPBs) [45].For example, various Bacillus-based formulations have been applied to crops such as tomato, canola, wheat, saffron, soybean, potato, apple, cassava, and tobacco, resulting in increased production and protection against pathogen attacks [46].However, our results also indicate that X. campestris and C. michiganensis promote root growth and development, potentially due to auxin and cytokinin production by these bacteria.Speculatively, this could be a strategy employed by the bacteria to expand the infection area of their hosts.However, in healthy plants, the induction of auxin and cytokinin responses is clearly beneficial considering the promotion of growth, including the root production to increase water and mineral absorption.
Arabidopsis root architecture changed in response to Bacillus treatment, evinced by the induction of lateral (secondary) roots.Plants produced lateral roots as a part of their natural growth and development, playing a crucial role in supporting the plant's overall structure, anchoring it to the substrate, and enhancing nutrient and water absorption.The production of lateral roots is regulated by auxins, such as indole-3-acetic acid (IAA), responsible for promoting their initiation, cell elongation, and differentiation.The differences in lateral root induction by Bacillus isolates may reflect the efficiency in the crosstalk with plant cells, yet to be investigated.Multiple PGPB produce auxins and cytokinins, enabling them to form a close association with plant roots by increasing the root surface and absorption area [47].In this study, we tested this phenomenon using reporter genes for auxin and cytokinin response.The results revealed a notable induction of the auxin reporter gene, indicating auxin accumulation following interaction with Bt R1, Bt R2, Bt tolworthi, Bt alesti, Bt kurstaki strain 1, and Bt kurstaki strain 2.Moreover, Bt R2, Bt kurstaki strain 2, and Bt entomocidus demonstrated a higher accumulation of cytokinins.A suitable crosstalk between auxin and cytokinin pathways is crucial for stem elongation, root development, and response to pathogen infections.In cases involving the interaction of R. solanacearum, high auxin accumulation was observed in leaf tissue, but plants displayed stunted development.This could potentially be a strategy employed by these bacteria to expand their infection area [48].Furthermore, the three phytopathogenic bacteria studied exhibited elevated levels of cytokinin accumulation, which may not be strictly necessary for the host but could facilitate the infection process.It has been reported that other Bacillus species produce phytohormones in vitro, including abscisic acid (ABA) and gibberellins (GA4 and GA3), which confer tolerance to both biotic and abiotic stress [49].
The successful use of beneficial microorganisms hinges on their adaptability to various agroecosystems, encompassing differences in soil, climate, and crop types.Thus, the application of native soil microorganisms to safeguard crops against phytopathogenic bacteria in agriculture holds promise.According to our findings, Bt strains can serve multiple purposes in intensive agriculture, from defending against pathogen attacks to promoting plant growth.This multifaceted approach has the potential to reduce the environmental impact of excessive agrochemical application.
B. thuringiensis exerts significant influences on plant growth through yet to be discovered induction of phytohormone synthesis such as auxins, gibberellins, and cytokinins, controlling plant growth, including cell elongation, root development, and overall biomass production.Additional research is needed to understand how Bacillus strains induce the expression of genes related to plant hormone signaling pathways to further influence the plant's physiological responses.This intricate interplay between Bacillus and phytohormones highlights the potential of these bacteria as biostimulants for promoting sustainable and robust plant growth in agricultural and horticultural settings.
Crop protection involving PGPB can complement modern biotechnological strategies within the framework of sustainable agriculture.This may include the use of genetically edited plants with reduced water consumption or increased drought tolerance via CRISPR/Cas9 gene editing, without compromising yield [50], or the engineering of plants to resist pathogen attacks, as seen in the case of Huanglongbing affecting citrus worldwide [51].Other options include the use of nanoparticles loaded with antimicrobials for controlling various phytopathogens [52].These alternatives should be considered part of integrated crop management.
The use of PCPB in sustainable agriculture is desirable because it is environmentally friendly; however, in a risk assessment of its use, the concern on the potential development of resistance in pathogens over time is possible.To consider a long-lasting use of such microorganisms, we should consider the selective pressure, the natural evolution of the pathogens to overcome competition with biocontrollers, the use of diverse combination of such PCPB to reduce resistance, the incorporation of different strategies to integrated pest-management (cultural practices, resistant crop varieties, and chemical control), and a regular monitoring of plant growth and pathogen population for timing adjustment.In summary, while the potential for resistance development exists, careful management practices, including the use of diverse biocontrol agents and integrated approaches, can help mitigate this risk and maintain the efficacy of biocontrol in sustainable agriculture.
We employed B. thuringiensis strains to enhance plant growth and provide protection against bacterial pathogens.Our study demonstrated that the inhibition of quorum sensing by these Bacillus strains is associated with increased plant vigor.The present research proposes the use of beneficial microorganisms as an environmentally friendly alternative.While the reduction of agrochemical use is desirable for promoting sustainable agriculture and enhancing crop yield, it presents new challenges.Potential future directions for research in this field could be focused in assessing its use under agricultural environments, optimizing the large-scale production of Bacillus-based products with stringent quality control, and their effective integration into plant management by producers worldwide.

Fig. 4
Fig.4 Effect of Bt strains on root system architecture in ARR5 Arabidopsis seedlings.A Petri dishes with the plant-bacteria interaction.Arabidopsis seedlings were inoculated with different Bt strains.Con-

Fig. 5
Fig. 5 Effect on auxin and cytokinin accumulation in Arabidopsis seedlings inoculated with Bt strains.A Representative images illustrating the impact of different Bacillus strains on the growth of primary roots, secondary roots, and shoots in Arabidopsis seedlings.Afterwards, seedlings were stained for GUS activity.B Arabidopsis

Table 1
Bacterial strains used in the present work *CDBB Collection of Microbial Strains and Cell Cultures.CNRF National Phytosanitary Reference Center.INECOL Institute of Ecology, A.C.

3
Quorum sensing inhibition evaluation in C. violaceum treated with Bt extracts.A